The purpose of this project is two fold: (1) to assess the regulation of reactive thiol(s) located in the cytoplasmic domain of the insulin receptor by agents that alter glutathione redox cycle; and (2) to perform fragmentation of thiol-biotinylated insulin receptors to determine the location of reactive thiol(s) within the receptor's sequence. Thiol-biotinylation by maleimidodibutyryl biocytin (MBB) was used as an index of insulin receptor thiol reactivity. Chinese hamster ovary cells transfected with the human insulin receptors (CHO/HIRc) were subjected to depletion of cellular glutathione level in the absence or presence of oxidative stress created by exogenous addition of hydrogen peroxide. We have found that under condition of cellular oxidation, depletion of glutathione led to a drastic loss of reactivity of the receptor thiol(s). Progress has been made in the isolation and characterization of a unique insulin receptor fragment containing thiol-biotinylated residue.